The T cell receptor (TCR)-CD3 complex is composed of a diverse TCR?? heterodimer noncovalently associated with the invariant CD3 dimers CD3??, CD3??, and CD3??. The TCR mediates peptide-MHC (pMHC) recognition, while the CD3 molecules transduce activation signals to the T cell. Whereas much is known about downstream T cell signaling pathways, the mechanism whereby TCR engagement by pMHC initiates signaling remains a mystery. Our goal is to understand the early events of TCR signaling by obtaining information on: 1) the spatial organization of the TCR-CD3 complex; and 2) possible allosteric changes in TCR conformation and/or dynamics upon binding pMHC that are relayed to CD3. These studies will be undertaken through a highly integrated and comprehensive project that combines multiple approaches, technologies, and investigators. To assure the generality of our findings, we will study both an MHC class I-restricted TCR (A6) and an MHC class II-restricted TCR (MS2-3C8). A6 recognizes a viral antigen from HTLV-1 bound to HLA-A2; MS2-3C8 recognizes a self-peptide from myelin basic protein bound to HLA-DR4. Our Specific Aims are: 1. Structure determination of the wild-type TCR-CD3 complex by NMR. Although the extracellular regions of CD3 are known to interact with the extracellular regions of the TCR, all attempts to crystallize TCR-CD3 complexes have been thwarted by the very low affinity of TCR-CD3 interactions in solution. We will employ NMR chemical shift perturbation and PRE to determine binding epitopes between CD3 and TCR. These data will be used to determine a structure for the wild-type TCR-CD3 complex. We will address whether pMHC binding alters TCR-CD3 interactions, possibly triggering T cell signaling. 2. Structural analysis of affinity- matured TCR-CD3 complexes by X-ray crystallography. We will attempt to overcome the weak association between TCR and CD3 ectodomains by in vitro directed evolution using yeast surface display to stabilize TCR-CD3 complexes for crystallization. Information on binding epitopes from NMR will be used to design TCR mutant libraries by focusing mutagenesis on those regions that contact CD3 (or vice versa). 3. Biological validation of the TCR-CD3 structure. We will validate TCR-CD3 interfaces identified by NMR or X-ray crystallography by evaluating the effects of structure-based mutations in TCR-CD3 interfaces on complex assembly, cell surface expression, signaling, and T cell function and development. The TCR-CD3 complex will be reconstituted using 2A-linked retroviral vectors to generate T cells or retrogenic mice expressing defined combinations of TCR and CD3 components. 4. Structural and dynamics analysis of free and pMHC-bound states of TCR?? ectodomains. We will address the allosteric change hypothesis by carrying out solution NMR analysis of full-length TCR ectodomains in free form and bound to pMHC, for both TCRs A6 and MS2- 3C8. We will investigate whether pMHC ligation induces changes in TCR conformation and/or dynamics and, if so, relate these changes to a possible mechanism for TCR triggering involving interactions with CD3.